﻿A new species and four new records of Bacidia (Lecanorales, Ramalinaceae) from South Korea, with a key to Korean species

﻿Abstract A new species, Bacidiafuscopallida Lee & Heo and four new records, B.ekmaniana R. C. Harris, Ladd & Lendemer, B.friesiana (Hepp) Körb., B.heterochroa (Müll. Arg.) Zahlbr. and B.suffusa (Fr.) A. Schneid., are described from South Korea. Bacidiafuscopallida differs from B.diffracta S. Ekman, the most similar species, by warted but non-granular thallus, paler and smaller apothecia without pruina, proper exciple without crystals, over 11-septate ascospores and smaller pycnidia and pycnoconidia. Bacidiaekmaniana is recorded new to Asia, B.heterochroa is reported new to northeastern Asia and B.friesiana and B.suffusa are new to Korea. Molecular analyses employing internal transcribed spacer (ITS) sequences strongly support the classification of the five species of Bacidia. A surrogate key is provided to assist in the identification of all 19 taxa in Bacidia of Korea.


Isolation, DNA extraction, amplification and sequencing
Hand-cut sections of ten to twenty ascomata per collected specimen were prepared for DNA isolation (Table 1) and DNA was extracted with a NucleoSpin Plant II Kit in line with the manufacturer's instructions (Macherey-Nagel, Düren, Germany). PCR amplifications for the internal transcribed spacer region (ITS1-5.8S-ITS2 rDNA) RNA genes were achieved using Bioneer's AccuPower PCR Premix (Bioneer, Daejeon, Korea) in 20-μl tubes with 16 μl of distilled water, 2 μl of DNA extracts and 2 μl of the primers ITS5 and ITS4 (White et al. 1990). The PCR thermal cycling parameters used were 95 °C (15 sec), followed by 35 cycles of 95 °C (45 sec), 54 °C (45 sec) and 72 °C (1 min) and a final extension at 72 °C (7 min), based on Ekman (2001). The annealing temperature was occasionally altered by ±1 degree in order to obtain a better result. PCR purification and DNA sequencing were accomplished by the genomic research company Macrogen (Seoul, Korea).

Phylogenetic analyses
An independent phylogenetic tree for the genus Bacidia was produced from 84 sequences from GenBank and 12 newly-generated sequences for the new species and the new records (Table 2). All ITS sequences were aligned and edited manually using ClustalW in Bioedit v.7.2.6.1 (Hall 1999). All missing and ambiguously aligned data and phylogenetically uninformative positions were removed and phylogenetically informative regions were finally analysed in MEGA X (Stecher et al. 2020). The final alignment comprised 930 bp, in which 102 variable regions were detected. The phylogenetically informative regions were 585. Phylogenetic trees with bootstrap values were obtained in RAxML GUI 2.0 beta (Edler et al. 2019) using the Maximum Likelihood method with a rapid bootstrap with 1,000 bootstrap replications and GTR GAMMA (GTR + G4) for the substitution matrix. The posterior probabilities were obtained in BEAST 2.6.4 (Bouckaert et al. 2019) using the GTR 123454 model, as the appropriate model of nucleotide substitution produced by the Bayesian model averaging methods with bModelTest (Bouckaert and Drummond 2017), empirical base frequencies, gamma for the site heterogeneity model, four categories for gamma and a 10,000,000 Markov Chain Monte Carlo chain length with a 10,000-echo state screening and 1,000 log parameters. Then, a consensus tree was constructed in TreeAnnotator 2.6.4 (Bouckaert et al. 2019) with the first 25% discard as a burn-in, no posterior probability limit, a maximum clade credibility tree for the target tree type and median node heights. All trees were displayed in FigTree 1.4.2 (Rambaut 2014) and edited in Microsoft Paint. Overall analyses in the materials and methods were undertaken based on Lee and Hur (2020).

Phylogenetic analyses
The new species is positioned in the genus Bacidia in the ITS tree (Fig. 2). The ITS tree describes B. fuscopallida, the new species, being nested with B. hostheleoides (Nyl.) Zahlbr., supported by a bootstrap value of 98 and a posterior probability of 1.00 for the branch. Bacidia fuscopallida is located in its own clade without any sequences close to it, although B. fuscopallida is sister to B. hostheleoides. Thallus corticolous, crustose, areoles in young stage and soon coarsely continuous or warted on aging, often overlapping for each other, rarely granular, thin when not overlapping, olivish-green, margin indeterminate, 40-90 μm thick; cortex indistinct, hyaline, up to 5 μm thick; medulla a little shown as mycelia below algal layer; photobiont chlorococcoid, cells globose to subglobose, 5-15 μm thick, algal layer composing most part of thallus, 35-80 μm thick. Prothallus indistinct or whitish-grey and endosubstratal when present.
Distribution and ecology. The species occurs on barks of Acer pictum var. mono, A. triflorum and Quercus mongolica. The species is currently known from the type collections.
Etymology. The species epithet indicates the pale brown colour of the lichen's apothecia.
Notes. The new species is similar to B. diffracta and B. polychroa (Th. Fr.) Körb. in having colourless epihymenium with pale orange-brown pigment and K+ purple reaction, distinctly pigmented hypothecium with yellow, orange or brown, long ascospores generally with L/W ratio over 10 amongst corticolous species. However, B. diffracta differs from the new species by granular thallus, darker and larger apothecia with pruina, proper exciple with radiating clusters of minute crystals, slightly wider ascospores with up to 11-septation and larger pycnidia and pynoconidia (Ekman 1996) (Table 3).
The new species is more similar to B. polychroa in having coarsely continuous or warted thallus. However, B. polychroa differs from the new species by greyish thallus, darker and larger apothecia often with pruina, proper exciple often with radiating clusters of minute crystals, wider ascospores and larger pycnidia and pycnoconidia (Ekman 1996;Smith et al. 2009) (Table 3).  Lendemer et al. (2016) The morphological and chemical characteristics of several species close to the new species are referenced from the previous literature. All information on the new species is produced from type specimens (KBA-L-0001010, KBA-L-0001011 and KBA-L-0001049) in this study. The new species is quite similar to B. purpurans R. C. Harris, Ladd & Lendemer in having greenish thallus with areoles and K+ purple reaction in epihymenium. However, B. purpurans differs from the new species by arachnoid prothallus, darker apothecia, green excipular rim adjacent to epihymenium, greyish epihymenium, shorter hypothecium, absence of crystals, larger ascospores and larger pycnidia and pycnoconidia (Lendemer et al. 2016) (Table 3).
The new species can be compared with B. hostheleoides in sharing non-pruinose apothecia and proper exciple without crystals. However, B. hostheleoides differs from the new species by greyish thallus, absence of prothallus, shorter hymenium, paler hypothecium and shorter ascospores with a few septa (Ekman 1996) (Table 3). Description. Thallus corticolous, crustose, somewhat granular when young and smoother when mature, grey, greenish-grey to pale grey, margin indeterminate. Prothallus generally not detected or whitish-grey when present.
Notes. Bacidia ekmaniana is easily confused with B. schweinitzii under the microscope, as well as in the field because both species often share their habitat and the habiti of both species look similar particularly when the ascomata of the latter are paler. Both species are often detected from one specimen under the microscope and those were frequently regarded as one species, i.e. B. schweinitzii. Generally, however, B. ekmaniana differs from the latter by paler ascomata. Bacidia ekmaniana has brown but not black apothecia when mature (Lendemer et al. 2016). Bacidia ekmaniana differs from the latter by colourless epihymenium and paler hypothecium as well.
Bacidia ekmaniana is more similar to B. arceutina than B. schweinitzii in morphology in having pale ascomata. However, B. ekmaniana differs from B. arceutina by the colourless to pale excipular rim, colourless epihymenium and wider ascospores with more septation (Ekman 1996; also see the key couplet 23). Bacidia ekmaniana is new to Asia and this is the second record after North America (Lendemer et al. 2016). Bacidia ekmaniana is supposed to occur widespread throughout the world as the species was assumed to be B. schweinitzii in the past. Phylogenetic analysis resulted in B. ekmaniana being located in its own clade in the genus Bacidia (Fig. 2). Description. Thallus corticolous, crustose, thin, little developed or indistinct, generally not continuous, minutely granular with contiguous granules when developed, pale grey with slightly brownish colour, margin indeterminate. Prothallus not detected.
Phylogenetic analysis resulted in B. friesiana of Korea (ON352609 and ON352610) being nested with the sequences of Russia (MH539765), supported by a bootstrap value of 100 and a posterior probability of 1.00 for the branch (Fig. 2). Bacidia friesiana was previously reported from Europe, North America and Russian Far East (Smith et al. 2009;Gerasimova et al. 2018). This is a new record to Korea. Specimens examined. South Korea, Gangwon Province, Yanggu, Nam-myeon, Dumu-ri, nearby a forested wetland, 38°02.12'N, 128°05.14'E, 421 m alt., on bark of Description. Thallus corticolous, crustose, continuous, wrinkled, or warted, pale yellowish-grey, margin indeterminate or determinate. Prothallus generally not present or locally present as blackish bordering a different lichen.
Chemistry. Epihymenium K+ purple or intensifying, extending to excipular rim. No lichen substance was detected by TLC.
Notes. Bacidia heterochroa is the most similar to B. laurocerasi (Delise ex Duby) Zahlbr. in having smooth thallus without granules, absence of crystals in exciple, epihymenium without green pigments, pale to colourless hypothecium, K+ purple in apothecial section and narrow ascospores less than 4 μm wide amongst corticolous species. However, B. heterochroa differs from B. laurocerasi by distinctly brown-pigmented paraphysial tips, less than 16-septate ascospores which are shorter but wider (less than 80 μm long but over 3.5 μm wide) and substrate preference to deciduous trees or shrubs (Ekman 1996;Brodo 2016; also see the key couplet 21).
Phylogenetic analysis resulted in B. heterochroa of Korea (ON352606, ON352612 and ON352613) being nested in a sister clade to B. laurocerasi, supported by a bootstrap value of 75 without a posterior probability as the Maximum Likelihood analysis did not match with the Bayesian Inference for the clade. The sequences of B. het-erochroa were not compared with previous records due to the lack of data (Fig. 2). Bacidia heterochroa was previously reported from Thailand in Asia (Aptroot et al. 2007) and this is a new record to northeastern Asia.
Notes. Bacidia suffusa is the most similar to B. russeola (Kremp.) Zahlbr. in having dark apothecia, generally colourless epihymenium without green pigment, long ascospores with the L/W ratio over 11, pale or colourless hypothecium and K+ purple reaction on epihymenium and nearby excipular rim amongst corticolous species. However, B. suffusa differs from B. russeola by the presence of pruina on the disc and in proper exciple as radiating clusters of crystals and more than 10-septate ascospores (Ekman 1996).
Phylogenetic analysis resulted in B. suffusa of Korea (ON352605, ON352614, ON352615 and ON352616) being nested in a sister clade of the sequences of Pakistan Key to the species of Bacidia in Korea (19 taxa) The key is composed of all 19 species in the genus Bacidia of Korea, including synonyms in Bacidina and Toniniopsis species.